To compare differences in chemical composition between plasma membrane and phagosome, or vacuolar, membrane in Acanthamoeba, we have developed methods for obtaining highly purified membrane fractions. Plasma membrane fractions are 42-fold purified over the homogenate and phagosome membrane fractions are estimated to have about 10% contaminating protein. Comparison of these two membranes on polyacrylamide gel electrophoresis showed significant quantitative differences in their composition. This result suggests that membrane exchange between surface and internal membrane systems is a complex process.